To observe the effect of immunofluorescence double staining on foamy macrophages and (MTB) in clinical paraffin-embedded tissue of tuberculous wound, and to compare with three routine staining methods. The experimental method was used. From April 2019 to May 2020, 10 patients with tuberculous wound (5 males and 5 females, aged 28-77 years) were treated in the Department of Burns and Plastic& Wound Repair Surgery of Xiang’an Hospital of Xiamen University. The paraffin tissue of the wounds were collected during extended debridement and preserved in the Department of Pathology of this hospital. Forty paraffin sections were made from the wound tissue of each patient. Hematoxylin-eosin (HE) staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining were performed respectively, with 10 sections in each method. The removal rate of sections in the four staining methods were calculated. The recognition and detection of wound granuloma tissue in the four staining methods were observed and counted, and the recognition and detection of foamy macrophages in the wound tissue of four staining methods was observed. The MTB detection, subtyping and distribution of foamy macrophages in the wound granuloma tissue and non-granuloma tissue in the four staining methods were compared. The detection rate of MTB in the wound granuloma tissue and non-granuloma tissue, the morphologic clarity of foamy macrophages, as well as the non-specific staining rate and the loss rate of positive reaction of MTB and foamy macrophages by Ziehl-Neelsen and immunohistochemical double staining were compared with those of immunofluorescence double staining. Data were statistically analyzed with Fisher’s exact probability test, one-way analysis of variance, independent sample -test and Wilcoxon signed rank test. The removal rates of sections of HE staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, and immunofluorescence double staining were 3% (3/100), 1% (1/100), 6% (6/100), and 2% (2/100), respectively. There was no statistically significant difference among the four groups (=0.256). All the four staining methods could identify granuloma tissue, and the number of granuloma structures was similar (F=1.284, =0.279). All the four staining methods were able to identify foamy macrophages in the wound tissue, which was detected in each section. No MTB was observed in the wound granuloma tissue or non-granuloma tissue by HE staining and immunohistochemical staining. MTB was observed distributing in the wound granuloma tissue and non-granuloma tissue by Ziehl-Neelsen and immunohistochemical double staining and immunofluorescence double staining, and most MTB was distributed in the wound granuloma tissue. Ziehl-Neelsen and immunohistochemical double staining could not distinguish foamy macrophages engulfed MTB from that non-engulfed MTB. Immunofluorescence double staining showed that foamy macrophages engulfed MTB mostly distributed in the wound granuloma tissue, and the foamy macrophages non-engulfed MTB mostly distributed in the wound non-granuloma tissue. The detection rates of MTB in wound granuloma and non-granuloma tissue in immunohistochemical double staining were (89.00±0.08)% and (82.67±0.05)%, respsctively, which were significantly higher than (54.56±0.14)% and (44.44±0.13)% in Ziehl-Neelsen and immunohistochemical double staining (t=-12.495, -7.961, <0.01). Compared with that of Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining showed better foamy macrophages clarity in wound tissue (Z=-3.162, P < 0.01). The nonspecific staining rate and positive reaction loss rate of MTB and foamy macrophages in wound tissue of immunofluorescence double staining were (9.11±0.07)% and (9.22±0.07)%, respectively, which were significantly lower than (20.67±0.06)% and (44±0.12)% of Ziehl-Neelsen and immunohistochemical double staining (t=4.569, 15.519, <0.01). Compared with HE staining, immunohistochemical staining, and Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining is clear, direct, and easy to operate. It can accurately realize co-location imaging of MTB and foamy macrophages in clinical paraffin-embedded tissue of tuberculous wound.

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